EP1080202A2 - Apport ameliore par la convection de vecteurs viraux adeno-associes (aav) - Google Patents

Apport ameliore par la convection de vecteurs viraux adeno-associes (aav)

Info

Publication number
EP1080202A2
EP1080202A2 EP99925906A EP99925906A EP1080202A2 EP 1080202 A2 EP1080202 A2 EP 1080202A2 EP 99925906 A EP99925906 A EP 99925906A EP 99925906 A EP99925906 A EP 99925906A EP 1080202 A2 EP1080202 A2 EP 1080202A2
Authority
EP
European Patent Office
Prior art keywords
aav
subject
virions
aadc
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP99925906A
Other languages
German (de)
English (en)
Other versions
EP1080202B1 (fr
Inventor
Krys Bankiewicz
Janet Cunningham
Jamie L. Eberling
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Avigen Inc
Original Assignee
University of California
Avigen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California, Avigen Inc filed Critical University of California
Priority to EP05017045A priority Critical patent/EP1621626B1/fr
Priority to EP05016619A priority patent/EP1621625B1/fr
Publication of EP1080202A2 publication Critical patent/EP1080202A2/fr
Application granted granted Critical
Publication of EP1080202B1 publication Critical patent/EP1080202B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01028—Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase

Definitions

  • the present invention relates generally to efficient delivery of viral vectors to the CNS. More particularly, the present invention relates to gene therapy for the treatment of central nervous system (CNS) disorders, particularly those disorders which involve the neurotransmitter dopamine.
  • CNS central nervous system
  • Parkinson's disease alone affects over 1 million people in the United States. Clinically, PD is characterized by a decrease in spontaneous movements, gait difficulty, postural instability, rigidity and tremor. Parkinson's disease is caused by the degeneration of the pigmented neurons in the substantia nigra of the brain, resulting in decreased dopamine availability. Altered dopamine metabolism has also been implicated in schizophrenic patients who show increased dopamine in certain areas of the brain.
  • CNS disorders such as PD are treated by systemic administration of a therapeutic agent.
  • Systemic administration is often inefficient because of a drug's inability to pass through the blood brain barrier and because many drugs cause peripheral side effects.
  • many potentially useful compounds, such as proteins cannot be administered systemically. If these compounds are successful in penetrating the blood-brain-barrier, they may also induce central nervous system side effects as well.
  • Treatment of PD currently involves oral administration of the dopamine-precursor, L-dopa often in combination with a compound such as carbidopa, a peripheral inhibitor of the enzyme aromatic amino acid decarboxylase (AADC) that decarboxylates dopa to dopamine.
  • AADC aromatic amino acid decarboxylase
  • Adeno-associated virus (AAV) systems are emerging as the leading candidates for use in gene therapy.
  • AAV is a helper-dependent DNA parvovirus which belongs to the genus Dependovirus.
  • AAV requires infection with an unrelated helper virus, either adenovirus, a herpesvirus or vaccinia, in order for a productive infection to occur.
  • the helper virus supplies accessory functions that are necessary for most steps in AAV replication.
  • AAV infects a broad range of tissue, and has not elicited the cyto toxic effects and adverse immune reactions in animal models that have been seen with other viral vectors, (see, e.g., Muzyczka, (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Flotte et al. (1993) PNAS USA 90:10613-10617; Kass- eiser et al. (1992) Gene Therapy 1 :395-402; Yange et al. PNAS USA 91 :4407- 4411 ; Conrad et al. (1996) Gene Therapy 3:658-668; Yang et al. (1996) Gene Therapy 3:137-144; Brynes et al. (1996) J.
  • AAV may be well adapted for delivering genes to the central nervous system (CNS).
  • CNS central nervous system
  • U.S. Patent Number 5,677,158 described methods of making AAV vectors.
  • AAV vectors containing therapeutic genes under the control of the cytomegalo virus (CMV) promoter have been shown to transduce mammalian brain and to have functional effects in models of disease.
  • AAV vectors carrying transgenes have been described, for example, in Kaplitt et al. (1994) Nature Genetics 8:148-153; WO 95/28493 published 26 October 1995; WO 95/34670, published 21 December 1995; During et al., (1998) Gene Therapy 5:820-827; Mandel et al.
  • AAV-CMV-TH was tested in a rodent model of Parkinson's Disease where it caused significant improvement in rotational behavior of 6- hydroxydopamine-lesioned rats (Fan et al. (1998) Human Gene Therapy 9:2527- 2537; Mandel et al. (1997) PNAS USA 94:14083-14088).
  • gene therapy of PD has focused on delivery of at least two genes encoding enzymes involved in dopamine synthesis, namely TH and AADC. These methods are subject to all of the delivery problems discussed above and, in addition, require that both genes are expressed in the proper amounts. Thus, treatment of PD using AAV-AADC in combination with L-dopa has also not been demonstrated.
  • the present invention provides methods for delivering recombinant AAV (rAAV) virions carrying a transgene to the central nervous system (CNS) of a subject, for example a human, using convection-enhanced delivery (CED).
  • CED can be conducted, for example, using either an osmotic pump or an infusion pump.
  • the transgene encodes an aromatic amino acid decarboxylase (AADC) or active fragment thereof.
  • AADC aromatic amino acid decarboxylase
  • the invention provides for methods for delivering recombinant AAV virions to a subject having a CNS disorder.
  • the rAAV virions encode a suitable therapeutic polypeptide and are administered into the CNS of the subject using CED.
  • the CNS disorder is Parkinson's disease (PD)
  • the rAAV virions are administered into the striatum of the CNS
  • the nucleic acid sequence encodes AADC.
  • methods for treating a neurodegenerative disease in a subject are provided.
  • a preparation of recombinant adeno-associated virus (rAAV) virions carrying a therapeutic nucleic acid sequence that is expressible in transduced cells is administered to the CNS using convection-enhanced delivery (CED).
  • the neurodegenerative disease is PD and the therapeutic polypeptide is an AADC.
  • the method of treating the neurodegenative disease also includes administering at least one additional therapeutic compound to the subject, for example, systemically administering L-dopa and, optionally, carbidopa.
  • methods of determining levels of dopamine activity in the CNS of subject are provided.
  • a labeled tracer is administered to the subject.
  • the tracer is preferably a compound that binds to a cell which utilizes dopamine and the label is preferably a radioisotope, for instance, 6-[ 18 F]-fluoro- L-m-tyrosine ( 18 F-FMT).
  • the detection of the label is indicative of dopamine activity via binding of the tracer.
  • the subject's CNS is imaged, for example using positron emission tomography (PET) scanning.
  • PET positron emission tomography
  • first and second therapeutic compounds in the manufacture of first and second medicaments for use in a method of treating a neurodegenerative disease, wherein the first medicament comprises recombinant AAV (rAAV) virions comprising a transgene encoding a therapeutic polypeptide, said first medicament administered to the subject by convection-enhanced delivery (CED), and wherein said second medicament comprises a therapeutic compound administered to the subject systemically is also provided.
  • rAAV recombinant AAV
  • CED convection-enhanced delivery
  • FIG. 1 panels a though d, depict dose responses (expression of the AAV-tk transgene) in rat following intracranial infusion pump delivery.
  • the tissue volume Figure la
  • mean area Figure lb
  • length Figure lc
  • number of cells Figure Id
  • Figure 2 is a half-tone reproduction showing labeling of rat brain tissue after injection of AAV vectors.
  • FIG. 3 panels a though d, depict intracranial delivery of AAV-tk through either an infusion pump (IP) or osmotic pump (OP).
  • IP infusion pump
  • OP osmotic pump
  • Figure 3d expressing the transgene are depicted.
  • Figures 4a, 4b, 4c and 4d are half-tone reproductions depicting CNS tissue infused with with vector carrying the tk transgene.
  • Figures 4a and b show expression of tk in neurons.
  • Figures 4 c and d show expression in neurons and glial near the site of osmotic pump infusion.
  • Figure 5 is a half-tone reproduction depicting Southern blot analysis of tissues from a subject infused with AAV-tk vector.
  • Figure 6 depicts immunostaining for AADC of the brains of MPTP- lesioned monkeys. The left side (control) shows limited staining, while the right side (AAV-AADC treated) shows broad AADC immunostaining.
  • Figure 7 is an FMT PET scan depicting dopamine activity in the brains of unilaterally MPTP-lesioned monkeys.
  • the left side shows limited activity on the lesioned side, while the right side (8 weeks post AAV-AADC administration) shows normal levels of dopamine activity.
  • panels A though C depict biochemical analysis of L-dopa levels in MPTP-lesioned monkeys.
  • Panel A shows that L-dopa is converted to dopamine by the AACD enzyme.
  • Striatum is AADC-rich, therefore, most of the L-dopa has been converted to dopamine in this region.
  • L-dopa conversion to dopamine is impaired and similar to cortical activity in AAV-LacZ treated monkeys. Both AAV-AADC-treated animals show almost normal rates of L- dopa to dopamine conversion.
  • Panel B depicts HVA analysis.
  • HVA is a metabolite of dopamine catabolism. Since cortical regions are not able to convert L-dopa to dopamine HVA levels are low.
  • striatum converts L-dopa to dopamine, therefore, dopamine is catabolised to HVA in this region.
  • AADC activity has not been restored in the AAV-LacZ-treated monkeys HVA levels in MTPT ipsilateral striatum are low.
  • HVA levels are significantly elevated in AAV-AADC-treated monkey in the MPTP ipsilateral striatum.
  • Panel C shows L-dopa levels were measured in the tissue punches following L-dopa administration.
  • FIG. 9 is a graph depicting activity of the AADC enzyme in- vitro.
  • AADC enzyme activity was determined by measuring rates of L-dopa to dopamine conversion. Cortical regions contain low levels of AADC. AADC activity in contralateral striatum is high, however it is variable since there is some dopaminergic lesion on that side of the brain. AADC activity in MPTP ipsilatral striatum is significantly reduced in AAV-Lac-Z-treated monkey while it is completely restored in the AAV-DDC monkeys.
  • Gene transfer or “gene delivery” refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene transfer provides a unique approach for the treatment of acquired and inherited diseases. A number of systems have been developed for gene transfer into mammalian cells. See, e.g., U.S. Patent No. 5,399,346.
  • vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • vector includes cloning and expression vehicles, as well as viral vectors.
  • recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
  • AAV virion is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat).
  • wt wild-type
  • AAV virus particle comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat.
  • single-stranded AAV nucleic acid molecules of either complementary sense, e.g., "sense” or “antisense” strands can be packaged into any one AAV virion and both strands are equally infectious.
  • a "recombinant AAV virion,” or “rAAV virion” is defined herein as an infectious, replication-defective virus composed of an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs.
  • a rAAV virion is produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring
  • Such techniques can be used to introduce one or more exogenous DNA moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
  • host cell denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in mo ⁇ hology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
  • a heterologous region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a cell transformed with a construct which is not normally present in the cell would be considered heterologous for pu ⁇ oses of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
  • a "coding sequence” or a sequence which "encodes” a particular protein is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • nucleic acid sequence refers to a DNA or RNA sequence.
  • the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl- methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter region is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • isolated when referring to a nucleotide sequence, is meant that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed or translated. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are know to those of skill in the art.
  • Two nucleic acid fragments are considered to "selectively hybridize" as described herein.
  • the degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules.
  • a partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
  • the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent” conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and SJ. Higgins, (1985) Oxford; Washington, DC; IRL Press).
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual. Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • aromatic amino acid decarboxylase or "AADC” refers to a polypeptide which decarboxylates dopa to dopamine.
  • AADC aromatic amino acid decarboxylase
  • a “functional homologue,” or a “functional equivalent” of a given polypeptide includes molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides which function in a manner similar to the reference molecule to achieve a desired result.
  • a functional homologue of AADC encompasses derivatives and analogues of those polyp eptides—including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino or carboxy termini thereof—so long as integrity of activity remains.
  • Techniques for determining nucleic acid and amino acid "sequence identity" or “homology” also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general,
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff,, Atlas of
  • sequence identity Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • Convection-enhanced delivery refers to any non-manual delivery of agents.
  • examples of convection-enhanced delivery (CED) of AAV can be achieved by infusion pumps or by osmotic pumps.
  • central nervous system includes all cells and tissue of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cereobrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like.
  • CSF cereobrospinal fluid
  • the "cranial cavity” refers to the area underneath the skull (cranium). Regions of the CNS have been associated with various behaviors and/or functions. For example, the basal ganglia of the brain has been associated with motor functions, particularly voluntary movement.
  • the basal ganglia is composed of six paired nuclei: the caudate nucleus, the putamen, the globus pallidus (or pallidum), the nucleus accumbens, the subthalamic nucleus and the substantia nigra.
  • the caudate nucleus and putamen although separated by the internal capsula, share cytoarchitechtomc, chemical and physiologic properties and are often referred to as the co ⁇ us striatum, or simply "the striatum.”
  • the substantia nigra which degenerates in Parkinson's patients, provides major dopaminergic input into the basal ganglia.
  • subject refers to a vertebrate, preferably a mammal.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals and pets.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • labeled tracer refers to any molecule which can be used to follow or detect a defined activity in vivo, for example, a preferred tracer is one that binds to cells that are utilizing dopamine. Preferably, the labeled tracer is one that can be viewed in a whole animal, for example, by positron emission tomograph (PET) scanning or other CNS imaging techniques. Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • Central to the present invention is the development of methods which allow for the efficient delivery of viral vectors, such as AAV, into the CNS of animal.
  • viral vectors such as AAV
  • researchers have had only minimal success delivering viral vectors to widespread areas of the brain.
  • convection-enhanced delivery devices for example, osmotic or infusion pumps
  • viral vectors can be delivered to many cells over large areas of the brain.
  • the delivered vectors efficiently express transgenes in CNS cells (e.g., neurons or glial cells).
  • novel gene therapy treatments for CNS disorders e.g., Parkinson's Disease
  • Parkinson's disease is treated by combining systemic L-dopa and/or carbidopa therapy with CNS-administration (e.g., via CED) of AAV vectors carrying a transgene encoding AADC, an enzyme involved in dopamine metabolism.
  • Advantages of the invention include, but are not limited to (i) efficient and widespread delivery of viral vectors (such as AAV) to the CNS; (ii) expression of nucleic acids (e.g., transgenes) carried by the viral vectors; (iii) identification of a therapeutic regime for Parkinson's Disease that involves delivery of one transgene in combination with administration of a pro-drug; and (iv) the ability to non-invasively monitor CNS gene therapy using PET scan.
  • viral vectors such as AAV
  • nucleic acids e.g., transgenes
  • viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology (see, for example, Ausubel or Maniatis, supra).
  • viral vectors carrying transgenes are assembled from polynuclotides encoding the transgene(s), suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
  • adeno-associated viral (AAV) vectors are employed.
  • a preferred method of obtaining the nucleotide components of the viral vector is PCR.
  • General procedures for PCR are taught in MacPherson et al.,
  • PCR A PRACTICAL APPROACH, (IRE Press at Oxford University Press, (1991)). PCR conditions for each application reaction may be empirically determined. A number of parameters influence the success of a reaction. Among these parameters are annealing temperature and time, extension time, Mg 2+ and ATP concentration, pH, and the relative concentration of primers, templates and deoxyribonucleotides. Exemplary primers are described below in the Examples. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • nucleotide sequences can be generated by digestion of appropriate vectors with suitable recognition restriction enzymes. The resulting fragments can then be ligated together as appropriate.
  • Polynucleotides are inserted into vector genomes using methods well known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and available in the art.
  • retroviral systems are known and generally employ packaging lines which have an integrated defective provirus (the "helper") that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence.
  • the cell line produces empty viral shells.
  • Producer lines can be derived from the packaging lines which, in addition to the helper, contain a viral vector which includes sequences required in cis for replication and packaging of the virus, known as the long terminal repeats (LTRs).
  • LTRs long terminal repeats
  • the gene of interest can be inserted in the vector and packaged in the viral shells synthesized by the retroviral helper.
  • the recombinant virus can then be isolated and delivered to a subject.
  • retroviral vectors include but are not limited to vectors such as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S. Patent No. 5,219,740, as well as derivatives of these vectors, such as the modified N2 vector described herein.
  • Retroviral vectors can be constructed using techniques well known in the art. See, e.g., U.S. Patent No 5,219,740; Mann et al. (1983) Cell 33:153-159.
  • Adenovirus based systems have been developed for gene delivery and are suitable for delivery according to the methods described herein.
  • Human adenoviruses are double-stranded DNA viruses which enter cells by receptor- mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro.
  • adenoviruses can infect human cells of hematopoietic, lymphoid and myeloid origin.
  • adenoviruses infect quiescent as well as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis.
  • adenovirus vectors have been developed which make use of these advantages.
  • the viral vectors are AAV vectors.
  • AAV vector is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
  • an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus.
  • the ITRs need not be the wild- type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region.
  • the control elements are selected to be functional in a mammalian cell.
  • the resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.
  • AAV ITRs adeno-associated virus inverted terminal repeats
  • AAV ITRs the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus.
  • AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence inte ⁇ osed between two flanking ITRs into a mammalian cell genome.
  • AAV ITR regions The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Berns, K.I. "Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.) for the AAV-2 sequence.
  • an "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
  • the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.
  • AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAVX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
  • Suitable DNA molecules for use in AAV vectors will be less than about 5 kilobases (kb) in size and will include, for example, a gene that encodes a protein that is defective or missing from a recipient subject or a gene that encodes a protein having a desired biological or therapeutic effect (e.g., an antibacterial, antiviral or antitumor function).
  • Preferred DNA molecules include those involved in dopamine metabolism, for example, AADC or TH.
  • AAV-AADC and AAV-TH vectors have been described, for example, in Bankiewicz et al. (1997) Exper't Neurol.
  • the selected nucleotide sequence such as AADC or another gene of interest, is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
  • control elements can comprise control sequences normally associated with the selected gene.
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a he ⁇ es simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • HSV he ⁇ es simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • synthetic promoters hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, CA).
  • heterologous promoters and other control elements such as CNS-specific and inducible promoters, enhancers and the like
  • heterologous promoters include the CMB promoter.
  • CNS-specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).
  • MBP myelin basic protein
  • GFAP glial fibrillary acid protein
  • NSE neuron specific enolase
  • inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia and aufin.
  • the AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • ORFs major AAV open reading frames
  • Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al. (1988) Molec. Cell. Biol.
  • AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra.
  • ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaCl, and either 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0oC (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation). Intermolecular "sticky end” ligations are usually performed at 30-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration).
  • AAV vectors which contain ITRs have been described in, e.g., U.S. Patent no. 5,139,941.
  • AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.
  • chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299: Jay et al. J. Biol. Chem. (1984) 259:6311.
  • an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M.R. (1980) Cell 22 ⁇ 19- 488), electroporation (Shigekawa et al.
  • suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) are preferred in the practice of the present invention.
  • the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral El a and Elb genes (Aiello et al. (1979) Virology 94:460).
  • the 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.
  • AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors.
  • AAV helper functions include one, or both of the major AAV
  • ORFs namely the rep and cap coding regions, or functional homologues thereof.
  • the Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters.
  • the Cap expression products supply necessary packaging functions.
  • AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.
  • AAV helper construct refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest.
  • AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither replicate nor package themselves.
  • AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945.
  • a number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Patent No. 5,139,941.
  • AAV rep coding region is meant the art-recognized region of the
  • AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome.
  • AAV rep coding region see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R.M. (1994) Human Gene Therapy 5:793-801.
  • Suitable homologues of the AAV rep coding region include the human he ⁇ esvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311).
  • AAV cap coding region is meant the art-recognized region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.
  • AAV cap coding region see, e.g., Muzyczka, N. and Kotin, R.M. (supra).
  • AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector.
  • AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection.
  • AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products.
  • Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers.
  • Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium.
  • selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Mo.). Other suitable markers are known to those of skill in the art.
  • the host cell (or packaging cell) must also be rendered capable of providing non AAV derived functions, or "accessory functions," in order to produce rAAV virions.
  • Accessory functions are non AAV derived viral and/or cellular functions upon which AAV is dependent for its replication.
  • accessory functions include at least those non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses. Particularly, accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art.
  • accessory functions are provided by infection of the host cells with an unrelated helper virus.
  • helper viruses include adenoviruses; he ⁇ esviruses such as he ⁇ es simplex virus types 1 and 2; and vaccinia viruses.
  • Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.
  • accessory functions can be provided using an accessory function vector.
  • Accessory function vectors include nucleotide sequences that provide one or more accessory functions.
  • An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell.
  • Accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid.
  • Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions. See, for example, International Publication No. WO 97/17548, published May 15, 1997.
  • Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art.
  • adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B.J. (1990) "Adeno- Associated Virus Helper Functions," in CRC Handbook ofParvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol. andlmmun. 158:97-129.
  • accessory functions are expressed which transactivate the AAV helper construct to produce AAV Rep and/or Cap proteins.
  • the Rep expression products excise the recombinant DNA (including the DNA of interest) from the AAV expression vector.
  • the Rep proteins also serve to duplicate the AAV genome.
  • the expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids.
  • productive AAV replication ensues, and the DNA is packaged into rAAV virions.
  • rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60oC for, e.g., 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile.
  • the resulting rAAV virions are then ready for use for DNA delivery to the CNS (e.g., cranial cavity) of the subject.
  • CNS e.g., cranial cavity
  • Methods of delivery of viral vectors include, but are not limited to, intra- arterial, intra-muscular, intravenous, intranasal and oral routes.
  • rAAV virions may be introduced into cells of the CNS using either in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cell will be removed from the subject, transduced with rAAV virions and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.
  • transduced cells can be transduced in vitro by combining recombinant AAV virions with CNS cells e.g., in appropriate media, and screening for those cells harboring the DNA of interest can be screened using conventional techniques such as Southern blots and/or PCR, or by using selectable markers.
  • Transduced cells can then be formulated into pharmaceutical compositions, described more fully below, and the composition introduced into the subject by various techniques, such as by grafting, intramuscular, intravenous, subcutaneous and intraperitoneal injection.
  • the rAAV virions will be formulated into pharmaceutical compositions and will generally be administered parenterally, e.g., by intramuscular injection directly into skeletal or cardiac muscle or by injection into the CNS.
  • conventional methods such as injection have not been shown to provide widespread delivery of viral vectors to the brain of the subject
  • central to the present invention is the discovery that viral vectors are efficiently delivered to the CNS via convection-enhanced delivery (CED) systems.
  • CED convection-enhanced delivery
  • the present inventors are the first to describe and demonstrate that CED can efficiently deliver viral vectors, e.g., AAV, over large regions of an animal's brain (e.g., striatum).
  • AAV vectors carrying reporter genes e.g., thymidine kinase (tk)
  • therapeutic genes e.g., AADC and tk
  • Any convection-enhanced delivery device may be appropriate for delivery of viral vectors.
  • the device is an osmotic pump or an infusion pump. Both osmotic and infusion pumps are commerically available from a variety of suppliers, for example Alzet Co ⁇ oration, Hamilton Co ⁇ oration, Alza, Inc., Palo Alto, California).
  • a viral vector is delivered via CED devices as follows.
  • a catheter, cannula or other injection device is inserted into CNS tissue in the chosen subject.
  • the striatum is a suitable area of the brain to target.
  • Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, MI. Positioning may also be conducted by using anatomical maps obtained by CT and/or MRI imaging of the subject's brain to help guide the injection device to the chosen target.
  • the methods described herein can be practiced such that relatively large areas of the brain take up the viral vectors, fewer infusion cannula are needed.
  • compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the protein of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit.
  • the pharmaceutical compositions will also contain a pharmaceutically acceptable excipient.
  • excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween ⁇ O, and liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • an effective amount of viral vector which must be added can be empirically determined. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • transgene could be expressed by the delivered viral vector.
  • separate vectors, each expressing one or more different transgenes can also be delivered to the CNS as described herein.
  • viral vectors delivered by the methods of the present invention be combined with other suitable compositions and therapies.
  • Parkinson's disease can be treated by co-administering an AAV vector expressing AADC into the CNS (e.g., into the caudate nucleus or putamen of the striatum) and additional agents, such as dopamine precursors (e.g., L-dopa), inhibitors of dopamine synthesis (e.g., carbidopa), inhibitors of dopamine catabolism (e.g., MaOB inhibitors), dopamine agonists or antagonists can be administered prior or subsequent to or simultaneously with the vector encoding AADC.
  • L-dopa and, optionally, carbidopa may be administered systemically.
  • AADC which is able to convert L-dopa into dopamine.
  • certain systemically-delivered compounds such as muristerone, ponasteron, tetracyline or aufin may be administered in order to regulate expression of the transgene.
  • Viral vectors expressing therapeutic transgenes can be used to treat various CNS disorders by providing therapeutic proteins or polypeptides.
  • the viral vectors are delivered to the CNS via the CED methods described herein as these methods provide the first effective way of broadly distributing viral vectors into the CNS.
  • disorders which may be treated include tumors, injury resulting from stroke and neurodegenerative diseases.
  • viral vectors which provide the enzyme AADC are used for the treatment of Parkinson's disease.
  • Parkinson's disease results from a selective loss of dopaminergic nigrostriatal neurons, resulting in a loss of input from the substantia nigra to the striatum.
  • Animal models of PD have been created, for instance by treating rats or primates with 6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells or by lesioning primates with the neurotoxin l-methyl-4-phenyl-l,2,3,4- tetrahydropyridine (MPTP), which produces a Parkinson's -like disease.
  • 6-OHDA 6-hydroxydopamine
  • MPTP neurotoxin l-methyl-4-phenyl-l,2,3,4- tetrahydropyridine
  • the present invention provides the first evidence that dopaminergic activity can be restored in Parkinson's patients (e.g., MPTP-lesioned monkeys), by administration of viral vectors carrying the transgene for AADC in combination with systemic (e.g., oral) administration of L-dopa and, optionally, carbidopa.
  • Parkinson's patients e.g., MPTP-lesioned monkeys
  • systemic e.g., oral
  • L-dopa e.g., oral
  • carbidopa e.g., carbidopa
  • AADC transgene
  • L-dopa the enzyme involved in the final step of dopamine biosynthesis, converting L-dopa to dopamine.
  • AADC-encoding nucleotides delivered by AAV vectors appear to be expressed mainly in the striatal neurons another important therapeutic advantage is the treatment's provision of a buffering mechanism for L-dopa.
  • Many side effects, such as dyskinesisas, are attributed to the inefficient buffering of Parkinsonian brain.
  • the methods described herein avoid this problem by allowing un-metabolized L-dopa to be stored in the neurons.
  • the delivery of AADC to the MPTP-treated striatum enables conversion of L-dopa to dopamine and the subsequent metabolism to DOPAC and HVA by striatal neurons.
  • striatal neurons can also store dopamine, since FMT was visualized in this region.
  • conversion rates of L-dopa to dopamine following AADC gene transfer were as robust and greater than seen in the normal striatum (see, e.g., Figure 7).
  • Parkinson's disease is an progressive disorder, it is not likely that an ongoing degeneration process will affect AADC expression in striatal neurons since they are not typically affected by idopathic Parkinson's disease.
  • the degeneration of the dopaminergic system in patients with idiopathic Parkinson's disease is not uniform.
  • the nigrostriatal pathway degenerates at much faster rate than mesolimbic pathway, leaving patients with an imbalance between the activity of the two pathways.
  • higher levels of L-dopa are needed to compensate for the degeneration of the nigrostriatal pathway, but this also results in increasingly higher dopamine levels in the nucleus accumbens and other parts of the mesolimbic system.
  • Such overstimulation may be responsible for some of the side effects associated with L-dopa treatment such as hallucinations.
  • the AAV-AADC vectors can be delivered by any suitable method, for example, injection, grafting, infusion, transplantation of cells carrying the vectors, etc.
  • the vectors are delivered by the CED methods described herein. As exemplified below, such delivery methods provide broad distribution and expression in CNS neurons and thereby provide a novel treatment regime for PD.
  • the present invention also provides methods of determining in vivo activity of an enzyme or other molecule. More specifically, a tracer which specifically tracks the targeted activity is selected and labeled. In a prefened embodiment, the tracer tracks dopamine activity, for example fluoro-L- - tyrosine (FMT) which binds to cells that utilize dopamine.
  • Suitable labels for the selected tracer include any composition detectable by spectroscopic, photochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include radiolabels (e.g., I8 F, 3 H, 125 1, 35 S, 32 P, etc), enzymes, colorimeteric labels, fluorescent dyes, and the like.
  • the label 18 F is used with FMT to quantify dopamine activity.
  • radiolabels may be detected using imaging techniques, photographic film or scintillation counters.
  • the label is detected in vivo in the brain of the subject by imaging techniques, for example positron emission tomography (PET). PET techniques are discussed in detail in Example 3 below.
  • PET positron emission tomography
  • the AAV-tk vector was constructed by placing the he ⁇ es simplex virus thymidine kinase (tk) gene under the transcriptional control of the cytomegalovirus (CMV) immediate early promoter in a pUC -based plasmid
  • a ⁇ -globin intron was located directly upstream from the tk gene and human growth hormone poly-A was placed downstream.
  • the entire cassette was flanked by AAV inverted terminal repeats (ITRs) that are required for gene expression, replication, and packaging into viral particles.
  • ITRs AAV inverted terminal repeats
  • Recombinant AAV virions were produced in human 293 cells (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) as follows.
  • the 293 cell line was cultured in complete DMEM (Biowhittaker) containing 4.5 g/liter glucose, 10% heat- inactivated fetal calf serum (FCS; Hyclone), and 2 mM glutamine.
  • FCS heat- inactivated fetal calf serum
  • Subconfluent 293 cells were co-transfected by calcium phosphate precipitation (see, e.g., Sambrook, et al.) with the AAV-tk expression cassette flanked by ITRs and helper plasmids derived from both AAV (pwl909, containing the AAV rep and cap genes) and adenovirus (pLadenol, containing E2a, E4, and adenoviral VA, and VA, RNA genes). After 6 hours, the media was changed to DMEM without serum and incubation was continued at 37°C in 5% CO 2 for 72 hours.
  • Tris buffer 100 mM Tris/150 mM NaCl, pH 8-0
  • the vector- containing pellet was solubilized in 50 mM Hepes Na/150 mM NaCl/25 mM EDTA, pH 8.0, and centrifuged at 10,000 x g for 15 minutes to pellet and remove insoluble material. Cesium chloride isopycnic gradient centrifugation was performed and
  • AAV-tk was recovered from the resulting gradient by isolating the fractions with in average density of 1.38 g/ml. PEG was again used to concentrate vector, which was then resuspended in 25 mM Hepes Na/150 mM NaCl, pH 7.4 and centrifuged as described to remove insoluble material. The stock was treated with DNAse and vector titer was determined by quantitative dot-blot hybridization.
  • EXAMPLE 2 IN VIVO DELIVERY OF AAV-TK: DOSAGES AND METHODS
  • the striatum was used to test dose response to the AAV vector because of its relatively large area of homogenous tissue and because it is a target for treatment of neurodegenerative disease and other central nervous system disorders.
  • rats were given 2.5 x 10 10 10 particles of AAV-tk by using the Harvard infusion pump (Harvard Apparatus Inc., Holliston, MA) or Alzet subcutaneous osmotic pumps (Alza Scientific Products, Palo Alto, CA).
  • Male Sprague-Dawley rats 250-300 g from Charles River Laboratories (Wilmington, MA) were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg, body weight) and prepped for surgery.
  • AAV-tk was continually administered to each rat. at a rate of 8 ⁇ l/h for 2.5 h using a Harvard infusion pump.
  • the loading chamber Teflon tubing 1/16th" OD x 0.03" ID
  • the infusion chamber (1/16" OD x 0.02" ID
  • subcutaneous osmotic pumps were used to deliver vector to one group of 6 animals.
  • AAV-tk was continually administered to each rat at a rate of 8 ⁇ l/hour for 24 h using Alzet osmotic pumps, model # 200 ID (ALZA Scientific Products, Palo Alto, CA).
  • the pump's reservoir and attached catheter (polyethylene 60 tubing) were filled with 2.5 x 10 10 particles of AAV-tk in a total volume of 200 ⁇ l artificial csf (Harvard Apparatus, Inc., Holliston, Mass.) Delivery was through a 27 gauge cannula fitted with fused silica.
  • the cannula was secured to the skull with a small stainless steel screw and dental cement, and the pump was implanted subcutaneously in the mid-scapular area of the back.
  • the surgical site was closed in anatomical layers with 9 mm wound clips. Twenty four hours later, pumps were removed by clipping and sealing the catheters but the implanted cannulas were left in place. Burr holes were filled with bone wax.
  • Tissues were immediately frozen in liquid N 2 and kept at -70°C until they were processed for genomic DNA.
  • PCR was performed using Perkin Elmer's GeneAmp PCR Core Kit and two 30-mer oligos derived from tk sequence (5'-AAGTCATCGGCTCGGGTACGTAGACGATATC-3' (SEQ ID NO:l) and 5' ATAGCAGCTACAATCCAGCTACCATTCTGC-3' (SEQ ID NO:2)) .
  • Reactions were performed in a PTC- 100 thermal cycler (MJ Reserach, Inc.) and resulted in a 158 bp per product in samples where vector was present.
  • Immunocytochemistry was used to detect transgene expression in every section that directly followed one stained with H & E. Thus, one out of every 12 sections was washed in PBS, treated with 3% H 2 O 2 for 30 m to block endogenous peroxidase activity, rinsed again in dH 2 0 and PBS, and incubated in blocking solution ( 10% goat serum + 0.01 % Triton-Xl 00 in PBS) for 30 m. Next, samples were incubated in polygonal anti-tk antibody (Yale ) (1:1000) for 1 h, washed three times in PBS, incubated in biotinylated goat anti-rabbit IgG (Vector) (1 :300) for 1 h, and washed again. Antibody binding was visualized with Streptavidin horseradish peroxidase (1:300) and VIP chromogen (Vector).
  • Transgene expression was quantitated for each brain by using a Ken-a- vision microprojector to project tk-immunostained sections onto an ARTZII graphic tablet.
  • the NTH image 1.6 program was used to capture and analyze images.
  • the total estimated number of positive cells for each brain was determined at a magnification of 100X using the following formula:
  • Infused tissue from animals receiving the highest titer demonstrated transgene expression in an average of 300 mm 3 of tissue (or approximately 60% of an adult rat cerebral hemisphere (Leyden et al. (1998) Behav. Brain Res. 87:59-67) as compared to volumes of 10 mm 3 for the middle-dose group and ⁇ 1 mm 3 for the low-dose group (Fig. la). Volumes were calculated from the mean areas and lengths of staining which both also showed significant differences between the groups (Fig. 1 b,c). Expression within a volume of transduced tissue was not uniform, however, but exhibited a gradient of staining.
  • Fig. Id illustrates that the total number of tk-positive cells in section, from the high dose group, estimated to average 169,000, is approximately 10X higher than that of the middle dose group.
  • PCR analysis was performed on 15 different organs and tissue from each of three rats who had received a high dose of vector. Regardless of the delivery method (infusion or osmotic pumps), a 458 bp PCR product from the tk gene could be detected in spinal cord, spleen, and both hemispheres of the brain using Southern blot analysis (Fig. 6). In one of the rats, vector sets were also detected in tissue from the kidney.
  • tissue damage was minimal, if present at all. There was no cellular infiltration, no necrosis in the needle tract, and minimal cortical necrosis in a few of the animals. Fresh bleeding was found in one of the high-dose rats, and hemosiderosis, indicating moderate bleeding in the past, was found in four of the high-dose animals. Alternatively, serious damage was noted in all of the animals in the osmotic pump delivery group including large necrotic areas swrounding the needle tract, cellular infiltrates, and hemosiderosis.
  • infusion of 2.5 x 10 10 particles of AAV-tk at 8 ⁇ l/h for 2.5 h is sufficient to partially distribute AAV vector to a volume of 300 mm 3 of tissue.
  • AAV AAV gene therapy vectors employing the CMV promoter are useful for treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. No expression was seen in mature glial cells, except in small areas of disturbed tissue where active gliosis was present.
  • AAV-CMV-tk vector is expressed well in glioma cells and, when given in conjunction with the prodrag ganciclovir, is effective in treating experimental gliomas in nude mice.
  • tk "suicide" gene is thought to be toxic to dividing cells, it should pose a risk only to the targeted tumor cells and not to surrounding neurons.
  • CMV promoter used in this study allows for strong transgene expression in neurons, the choice of cell-type-specific promoters will allow targeting of AAV to other CNS components such as oligodendrocytes and glial calls.
  • AAV delivered to brain is contained mostly in the central nervous system. Others have demonstrated retrograde transport of viruses between the two hemispheres of brain and ability of viruses to reach spinal cord via circulating cerebral spinal fluid. The appearance of vector in the spleen is curious, and suggests a couple of mechanisms. One is that virus enters the bloodstream during the infusion process and circulated through the spleen where it is "scavenged". If this were the case, however, other tissues that have been shown to be inducible by AAV would be expected to also take up virus. Another possible mechanism could be one exhibited by dendritic cells.
  • AAV Alzheimer's disease
  • EXAMPLE 3 GENE THERAPY OF PARKINSON'S DISEASE Convection-enhanced delivery of AAV vectors carrying the transgene encoding AADC was shown to restore dopaminergic systems in MPTP-induced Parkinson's disease in monkeys as follows.
  • MPTP is it converted in the C ⁇ S to MPP+ by monoamine oxidase B.
  • MPP+ is a potent neurotoxin which causes degeneration of the nigral dopaminergic neurons and loss of the nigro-striatal dopamine pathway, as seen in Parkinson's disease.
  • MPTP-lesioned animals were clinically evaluated once a week using a clinical rating scale and activity monitoring for 5 months prior to surgery. Following MPTP administration, the animals developed clinical signs of
  • Parkinson's disease manifested by general slowness, bradykinesia, rigidity, balance disturbances, and flexed posture.
  • the left arm was less frequently used than the right in all of the monkeys, and all showed signs of tremor.
  • all of the monkeys had moderate to severe stable parkinsonian scores (23+ 1.7, 23+1.2, 24+1.7, 19+3) during the 5 month post- MPTP period.
  • the expression cassette contains a CMV promoter, a chimeric intron composed of a CMV splice donor and a human ⁇ - globin splice acceptor site, human growth hormone polyadenylation sequence, and flanking AAV ITRs (inverted terminal repeats) (Herzog, R.W., et al. (1999) Nature Medicine 5:56-63.).
  • the vector pAAV-LacZ was constructed as follows. The AAV coding region of pSub201 (Samulski et al. (1987) J. Virol 61:3096-3101), between the Xbal sites, was replaced with EcoRI linkers, resulting in plasmid pAS203. The EcoRI to Hindlll fragment of pCMV ⁇ (CLONETECH) was rendered blunt ended and cloned in the Klenow treated EcoRI site of pAS203 to yield pAAV-lacZ.
  • Plasmid HI 9 encodes a modified AAV-2 genome designed to enhanced AAV vector production while suppressing the generation of replication competent pseudo-wild type virus.
  • the plasmid contains a P5 promoter moved to a position 3' of the cap gene and the promoter is replaced by a 5' untranslated region primarily composed of a FLP recombinase recognition sequence. pH19 was constructed so as to eliminate any regions of homology between the 3' and 5' ends of the AAV genome. Additionally, the seven base pair TATA box of the pH19 P5 promoter was destroyed by mutation of that sequence to GGGGGGG. pH19 was constructed in a several step process using AAV-2 sequences derived from the AAV-2 provirus, pSM620.
  • pSM620 was digested with Smal and PvuII, and the 4543 bp, rep and cap gene encoding Smal fragment was cloned into the Smal site of pUCl 19 to produce the 7705 bp plasmid, pUCrepcap.
  • the remaining ITR sequences flanking the rep and cap genes were then deleted by oligonucleotide-directed mutagenesis using the following oligonucleotides:
  • the resulting plasmid, pUCRepCapMutated (pUCRCM) (7559 bp) contains the entire AAV-2 genome without any ITR sequence (4389 bp). Srfl sites, in part introduced by the mutagenic oligonucleotides, flank the rep and cap genes in this construct.
  • the AAV sequences correspond to AAV-2 positions 146- 4,534.
  • pUCRCM was mutagenized with primer P547 (5'-GGT TTG AAC GAG CGC TCG CCA TOGS') (SEQ ID NO:5).
  • P547 5'-GGT TTG AAC GAG CGC TCG CCA TOGS'
  • pUCRCM47III The resulting 7559 bp plasmid was called pUCRCM47III.
  • the polylinker of pBSIIsk+ was changed by excision of the original with BSSHII and replacement with oligonucleotides blunt 1 and 2.
  • the resulting plasmid, bluntscript is 2830 bp in length and the new polylinker encodes the restriction sites EcoRV, Hpal, Srfl, Pmel, and Eco47III.
  • the blunt 1 and 2 sequences are as follows: blunt 1; 5'-CGC GCC GAT ATC GTT AAC GCC CGG GCG TTT AAA CAG
  • TAT CGG-3' (SEQ ID NO:7) pHl was constructed by ligating the 4398 bp, rep and cap gene encoding
  • pHl is 7228 bp in length.
  • pH2 is identical to pHl except that the P5 promoter of pHl is replaced by the 5' untranslated region of pGN1909. To do this, the 329 bp Ascl(blunt)-Sfil fragment encoding the 5' untranslated region from pW19091acZ was ligated into the 6831 bp Smal(partial)-Sfil fragment of pHl creating pH2.
  • pH2 is 7156 bp in length.
  • a P5 promoter was added to the 3' end of pH2 by insertion of the 172 bp
  • SmaI-Eco43III fragment encoding the p5 promoter from pUCRCM47III into the Eco47III site in pH2. This fragment was oriented such that the direction of transcription of all three AAV promoters are the same. This construct is 7327 bp in length.
  • the TATA box of the 3' P5 (AAV-2 positions 255-261, sequence
  • TATTTAA was eliminated by changing the sequence to GGGGGGG using the mutagenic oligonucleotide 5DIVE2 (5'-TGT GGT CAC GCT GGG GGG GGG
  • Pladeno 5 is a plasmid that provides a complete set of adenovirus helper functions for AAV vector production when transfected into 293 cells. Essentially, it is composed of the E2A, E4, and VA RNA regions from adenovirus-2 and a plasmid back bone. The plasmid was constructed as follows.
  • pBSIIs/k+ was modified to replace the 637 bp region encoding the polylinker and alpha complementation cassette with a single EcoRV site using oligonucleotide directed mutagenesis and the following oligonucleotide: 5'-CCG CTA CAG GGC GCG ATA TCA GCT CAC TCA A-3' (SEQ ID NO:9).
  • a polylinker encoding the restriction sites BamHI, Kpnl, Srfl, Xbal, C , Bstl 1071, Sail, Pmel, and Ndel was then cloned into the EcoRV site (5'-GGA TCC GGT ACC GCC CGG GCT CTA GAA TCG ATG TAT ACG TCG ACG TTT AAA CCA TAT G-3') (SEQ ID NO: 10).
  • Adenovirus-2 DNA was digested and restriction fragments encoding the
  • E2A region (a 5,335 bp, Kpnl-Srfl fragment corresponding to positions 22,233- 27,568 of the adenovirus-2 genome) and the VA RNAs (a 731 bp, EcoRV-SacII fragment corresponding to positions 10,426-11,157 of the adenovirus-2 genome) were isolated.
  • the E2A fragment was installed between the Sail and Kpnl sites of the polylinker.
  • An E4 region was first assembled in pBSIIs/k+ by ligating a 13,864 bp, BamHI- Avrll fragment corresponding to adenovirus-2 positions 21,606-35,470 (encoding the 5' end of the gene) and a 462 bp, Avrll and Srfl, digested PCR fragment corresponding to adenovirus-2 positions 35,371-35,833 (encoding the 3' end of the gene) between the BamHI and Smal sites of pBSIIs/k+.
  • the oligonucleotides used to produce the PCR fragment were designed to introduce a Srfl site at the junction were the E4 promoter and the adenovirus terminal repeat intersect and have the sequences 5'-AGA GGC CCG GGC GTT TTA GGG CGG AGT AAC TTG C-3' (SEQ ID NO:l l)and 5'-ACA TAC CCG CAG GCG TAG AGA C-3' (SEQ ID NO: 12).
  • the intact E4 region was excised by cleavage with Srfl and Spel and the 3,189 bp fragment corresponding to adenovirus-2 positions 32,644-35,833 was cloned into the E2A intermediate between the Srfl and Xbal sites.
  • VA RNA fragment was inserted into the Bstl 107 site after T4 polymerase-mediated blunt end modification of the SacII site.
  • the genes in pladeno 5 are arranged such that the 5' ends of the E2A and E4 promoters abut, causing the regions to transcribe away from each other in opposite directions.
  • the VA RNA genes which are located at the three prime end of the E4 gene, transcribe towards the E4 gene.
  • the plasmid is 11,619 bp in length.
  • the HEK 293 cell line (Graham, F.L., Smiley, J., Russel, W.C., and Naiva, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36:59-72.) was cultured in complete DMEM (BioWhittaker) containing 4.5 g/liter glucose, 10% heat-inactivated fetal calf serum (FCS), and 2mM glutamine at 37°C in 5%CO 2 in air. Forty T225 flasks were seeded with 2.5xl0 6 cells each and grown for three days prior to transfection to 70-80% confluency (approximately 1.5xl0 7 cells per flask).
  • the vector production process involved co-transfection of HEK 293 cells with 20 ⁇ g of each of the following three plasmids per flask: the AAV-AADC plasmid, the AAV helper plasmid (pHLP19, containing the AAV rep and cap genes), and the adenovirus helper plasmid (pladeno-5, previously known as pVAE2AE4-2 (4) and composed of the E2A, E4, and VA RNA genes derived from purified adenovirus-2), using the calcium phosphate method (Wigler, M. et al. (1980) Transformation of mammalian cells with an amplifiable dominant-acting gene. Proc. Natl. Acad. Sci.
  • the resulting precipitate was removed by centrifugation (10,000 g for 15 min.) and discarded. The supernatant was then made 10% in PEG(8000), and was placed on ice for 3 hrs. The precipitate was collected by centrifugation (3000 g for 30 min) and resuspended in 4 ml of 50 mM NaHEPES, 0.15M NaCl, 25 mM EDTA (pH 8.0) per 20 T225 flasks. Solid CsCl was added to produce a density of 1.4 g/ml and the sample was centrifuged at 150,000 g for 24 hrs in a Beckman TI70 rotor.
  • AAV- containing fractions were pooled, adjusted to a density of 1.4 g/ml CsCl, and centrifuged at 350,000 g for 16 hrs in a Beckman NVT65 rotor. The fractions containing AAV were then concentrated and diafiltered against excipient buffer (5% sorbitol in PBS). The titer of the purified AAV-AADC vector was determined using quantitative dot blot analysis and vector stocks were stored at -80°C.
  • Infusion cannulae were flushed with saline to assess the integrity between the needle and tubing interface.
  • Sterile infusion cannulae and loading lines were connected using the appropriate fittings with extreme caution taken to prevent the collection of air bubbles in the system.
  • Non-sterile oil infusion lines were prepared as previously described and 1 ml gas tight Hamilton syringes filled with oil were attached to a Harvard infusion pump.
  • Six infusion cannulae were fitted onto microdialysis holders (3 cannulae per holder) and mounted onto a stereotactic tower.
  • the needle cannulae were primed with AAV and the infusion system transferred to the surgery table.
  • Initial infusion rates were set at 0.1 pl/min., the lines visually inspected to ensure a smooth flow of fluid through the system, and the cannulae manually lowered to their target sites. A final visual inspection was performed to check for any air bubbles in the infusion system.
  • the cannula system consisted of three components: (i) a sterile infusion cannula; (ii) a sterile loading line housing AAV-AADC or AAV-LacZ (control); and (iii) a non-sterile infusion line containing olive oil. Preparation of each line is described here briefly.
  • the infusion cannula consisted of 27 G needles (outer diameter, .03"; inner diameter, .06”; Terumo Co ⁇ ., Elkton, MD) fitted with fused silica (outer diameter, .016", inner diameter, .008"; Polymicro Technologies, Phoenix, AZ), and placed in Teflon tubing (.03" ID, Upchurch Scientific, Seattle, WA) such that the distal tip of the silica extended approximately 15 mm out of the tubing.
  • the needle was secured into the tubing using superglue and the system was checked for leaks prior to use.
  • a Tefzel fitting and ferrule were attached to connect the adjacent loading line.
  • Loading and infusion lines consisted of 50 cm sections of Teflon tubing (outer diameter, .062"; inner diameter, .03") fitted with Tefzel 1/1 6" ferrules, unions, and male Luer-lock adapters (Upchurch Scientific, Oak Harbor, WA) at the distal ends.
  • the sterile loading lines accommodated up to a 1000 ml volume and were primed with saline prior to use.
  • Ketamine Ketaset; 1 0 mg/kg, i.m.
  • a venous line was established using a 22 gauge catheter positioned in the cephalic or saphenous vein to deliver isotonic fluids at 5-10 ml/kg/hr.
  • Isoflurane (Aerrane, Omeda PPD Inc., Liberty, NJ) was delivered at 1-3% until the animal maintained a stable plane of anesthesia.
  • the head was placed in an MRI compatible stereotactic frame according to pre-set values attained during a baseline MRI scan.
  • the animal was instrumented with subcutaneous electrocardiogram electrodes, a rectal probe and the body covered with circulating water blankets to maintain a core temperature of 36-38 °C. Electrocardiogram and heart rate (using the Silogic ECG-60, Stewartstown, PA) and body temperature were continuously monitored during the procedure. The head was prepped with Betadine and 70% ethanol, a sterile field was created and a midline incision performed through the skin, muscle and fascia using electrocautery (Surgistat Electrosurgery, Valleylab Inc., Boulder, CO).
  • the cannulae assembly was raised at a rate of 1 mm/min. until it was out of the cortex.
  • the cortex was rinsed with saline, the bone margins trimmed with ronguers and the wound site closed in anatomical layers.
  • Analgesics Nao ⁇ han, IM
  • antibiotics Flocillin, IM
  • Animals were monitored for full recovery from anesthesia, placed in their home cages and clinically observed (2x/day) for approximately five days following surgery. Total neurosurgery time was 4.5 hours per animal.
  • mice were assessed for any signs of abnormal behavior. Animals were observed and rated by the veterinary technicians twice a day using clinical observation forms. All monkeys recovered from the surgery within 2 hours and were able to maintain themselves, including feeding and grooming. There were no signs of any adverse effects during the entire 8-week post-surgical period.
  • Magnetic resonance imaging Visualization of the target site is crucial for the precise placement of cells within the caudate nucleus or putamen.
  • Stereotactic procedures combined with MRI were used in order to accurately place the cannula within the desired targeted structures. All animals were scanned before surgery to generate accurate stereotactic coordinates of the target implant sites for each individual animal. The same fiducial markers that are used for PET scanning were placed on the frame for co-registration of MRI and PET images. Briefly, during the scanning procedure, the animals were sedated using a mixture of ketamine (Ketaset, 7 mg/kg, im) and xylazine (Rompun, 3 mg/kg, im).
  • the animals were placed in an MRI compatible stereotactic frame, earbar and eyebar measurements were recorded, and an IV line was established.
  • TR repetition time
  • TE echo time
  • the field of view was 15 cm, with a 192 matrix and a 2 NEX (number of averages per signal information). Baseline scanning time was approximately 20 minutes.
  • Rostro-caudal and medio-lateral distribution of a targeted structure was determined using the coronal MR images. Surgical coordinates were determined from magnified coronal images (1.5x) of the caudate nucleus and putamen.
  • PET Positron Emission Tomography
  • the monkeys were then injected with 10-1 5 mCi of the AADC tracer, 6-[ 18 F]fluro-L- -tyrosine (FMT) and imaging began. Imaging continued for 60 min, at which time the monkey was repositioned so as to image a second slice 6 mm caudal to the first.
  • FMT fluro-L- -tyrosine
  • the PET and MR datasets were co-registered and regions of interest (ROs) were drawn for the striatum in the contralateral hemisphere (the side opposite to ICA MPTP infusion) on PET data collected at 50 to 60 min (slice 1) and from 65 to 75 min (slice 2) with reference to the MR.
  • ROI 1 regions of interest
  • SAO regions of interest
  • Mirror images of the ROs were created in the ipsilateral hemisphere (side of MPTP infusion) and radioactivity counts (cmVsec) were determined for each ROI. Striatal counts were averaged over the two slices for each study.
  • FMT uptake asymmetry ratios were calculated for each animal at each time point by subtracting the counts for the ipsilateral (lesioned) striatum from the counts for the contralateral (un- lesioned) striatum and dividing by the average counts for the ipsilateral and contralateral striata.
  • a change score was calculated by subtracting the asymmetry ratio from the second PET study from the asymmetry ratio for the baseline study for each animal. Unpaired t-tests were used to compare the change in pet asymmetry ratios for the AAV-AADC and AAV- LacZ monkeys.
  • the antibody binding was visualized with streptavidin horseradish peroxidase (Vector Labs, 1 :300) and DAB chromogen with nickel (Vector Labs). Sections were then coverslipped and examined under a light microscope. Following tissue punching the fresh-frozen blocks were sectioned at 20 um. Sections were stained with H&E and for DDC-IR.
  • Quantitative estimates of the total number of AAV-infected cells within the caudate nucleus, putamen and globus pallidus were determined by using an optical dissector procedure.
  • the optical dissector system consisted of a computer assisted image analysis system, including an Leitz Otholux 11 microscope hard- coupled to a Prior H128 computer-controlled x-y-z motorized stage, a high sensitivity Sony 3CCD video camera system (Sony, Japan) and a Macintosh G-3 computer. All analyses were performed using NeuroZoom software (La Jolla, CA). Prior to each series of measurements, the instrument was calibrated.
  • the region of positive neurons in the caudate, putamen and globus pallidus was outlined at low magnification (2.5x objective). Because of the diffuse presence of AAV-infected cells within the striatum, 1% of the outlined region was measured with a systematic random design of dissector counting frames (1 505 1IM2) using a 63x plan-neofluar immersion objective with a 0.95 numerical aperture. Based on pilot experiments at least four sections equally spaced were sampled. By using the dissector principle, up to 200 AADC positive neurons were sampled by optical scanning by using uniform, systematic and random design procedures for all measurements. The average thickness of the sections was measured at 23 microns.
  • TH-IR staining revealed robust reduction of the nigrostriatal fibers and cell bodies in the substantia nigra on the ipsilateral side in all of the monkeys.
  • the contralateral side showed variable reductions of TH and AADC-IR in the striatum and the substantia nigra.
  • DDC-IR paralleled TH-IR only in the monkeys treated with AAV-LacZ.
  • the AAVAADC-treated monkeys showed robust AADC staining on the ipsilateral side that exceeded staining seen on the contralateral side.
  • a high density of AADC-TR cells was seen throughout 80% of the striatum and 100% of the globus pallidus in one of the AAV-AADC treated animals.
  • AADC-IR cells were found in over 60% of the ipsilateral striatum, with 7,515 cells per mm 3 in the caudate and 15,352 cells per mm 3 in the putamen and 3,850 cells per mm 3 in the globus pallidus. No AADC cells were found in the contralateral striatum.
  • AAV-infected cells were positive for the neuronal marker, Neu-N.
  • AAV-AADC-treated monkeys one out of 4-6Neu-N-positive cells was AADC-positive in the caudate and putamen, and one out of 3-4 Neu-N-positive cells was AADC positive in the globus pallidus. None of the AAV-infected cells in the Lac-Z or AADC-treated monkeys were GFAP -positive.
  • Brain regions were removed from fresh frozen blocks using a micropuncher in order to evaluate tissue levels of L-dopa and dopamine metabolites and the activity of AADC and the presence of the AAV- vector. Brain regions included striatum and cortex.
  • Frozen micropunches were collected, and homogenized by ultrasonic processing in 300 pi of 0.1 M perchloric acid (Fisher Scientific) containing 1 % ethanol, and 0.02% EDTA (Fisher Scientific). Fifty pi of the homogenate was removed for protein analysis (BCA Protein Assay Kit Pierce #23225), and the remainder centrifuged in a mirocentrifuge for 1.5 minutes at maximum speed.
  • the detector output was recorded and analyzed with the Waters Millennium 32 Chromatography Manager.
  • L-dopa As expected, there was no decarboxylation of L-dopa to dopamine within the cortex, however, in the striatum on the side contralateral to MPTP administration, L-dopa was converted to dopamine and further metabolized to HVA. In the MPTP-treated striatum of the AAV-Lac-Z monkeys , L-dopa was not converted to dopamine, nor was it metabolized to HVA. Tissue levels of L-dopa also remained at the same levels as in the cortex in AAV-Lac-Z treated monkeys. In the MPTP-treated striatum of AAV-AADC-treated monkeys, L-dopa was converted to dopamine and HVA and tissue levels of L-dopa in this region were reduced.
  • AADC activity was very low in the cortical regions and in the MPTP- treated striatum of AAV-LacZ-treated monkeys. L-dopa was converted to dopamine in the contralateral striatum, suggesting high levels of AADC activity.
  • the tissue punches from MPTP-treated striatum of AAV-AADC infected monkeys contained extremely high dopamine levels with only traces of L-dopa left.
  • the invention provides a novel and efficient treatment method for CNS disorders, such as Parkinson's Disease.
  • the invention also provides methods for determining dopamine activity in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Mycology (AREA)
  • General Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Psychology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
EP99925906A 1998-05-27 1999-05-26 Apport par la convection de vecteurs viraux adeno-associes (aav) codant pour l'aadc Expired - Lifetime EP1080202B1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP05017045A EP1621626B1 (fr) 1998-05-27 1999-05-26 Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
EP05016619A EP1621625B1 (fr) 1998-05-27 1999-05-26 Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US8694998P 1998-05-27 1998-05-27
US86949P 1998-05-27
US13474899P 1999-05-18 1999-05-18
US134748P 1999-05-18
PCT/US1999/011687 WO1999061066A2 (fr) 1998-05-27 1999-05-26 Apport ameliore par la convection de vecteurs viraux adeno-associes (aav)

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP05017045A Division EP1621626B1 (fr) 1998-05-27 1999-05-26 Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
EP05016619A Division EP1621625B1 (fr) 1998-05-27 1999-05-26 Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection

Publications (2)

Publication Number Publication Date
EP1080202A2 true EP1080202A2 (fr) 2001-03-07
EP1080202B1 EP1080202B1 (fr) 2006-01-25

Family

ID=26775343

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99925906A Expired - Lifetime EP1080202B1 (fr) 1998-05-27 1999-05-26 Apport par la convection de vecteurs viraux adeno-associes (aav) codant pour l'aadc

Country Status (9)

Country Link
US (7) US6309634B1 (fr)
EP (1) EP1080202B1 (fr)
JP (2) JP2002516295A (fr)
AT (3) ATE435917T1 (fr)
CA (1) CA2329259C (fr)
DE (3) DE69929600T2 (fr)
ES (3) ES2324540T3 (fr)
PT (1) PT1080202E (fr)
WO (1) WO1999061066A2 (fr)

Families Citing this family (122)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011244A2 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
ES2324540T3 (es) * 1998-05-27 2009-08-10 Genzyme Corporation Vectores aav para la fabricacion de medicamentos para la administracion potenciada por conveccion.
ATE402254T1 (de) * 1998-05-28 2008-08-15 Us Gov Health & Human Serv Aav5 vektoren und deren verwendung
US6855314B1 (en) * 2000-03-22 2005-02-15 The United States Of America As Represented By The Department Of Health And Human Services AAV5 vector for transducing brain cells and lung cells
US7672712B2 (en) * 2000-03-30 2010-03-02 The Regents Of The University Of Michigan Internal marker device for identification of biological substances
US7098374B2 (en) 2001-02-09 2006-08-29 University Of Florida Research Foundation, Inc. Human disease modeling using somatic gene transfer
US7588757B2 (en) * 2001-03-14 2009-09-15 Genzyme Corporation Methods of treating Parkinson's disease using recombinant adeno-associated virus virions
US7182944B2 (en) * 2001-04-25 2007-02-27 The United States Of America As Represented By The Department Of Health And Human Services Methods of increasing distribution of nucleic acids
US20050191638A1 (en) * 2002-02-20 2005-09-01 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20030050273A1 (en) * 2001-08-29 2003-03-13 Keiya Ozawa Compositions and methods for treating neurodegenerative diseases
WO2003037260A2 (fr) * 2001-10-30 2003-05-08 Biogen, Inc. Methodes et compositions pour le traitement de la maladie de parkinson
US7419817B2 (en) * 2002-05-17 2008-09-02 The United States Of America As Represented By The Secretary Department Of Health And Human Services, Nih. Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography
US7090836B2 (en) 2002-06-21 2006-08-15 Institut Pasteur Vector for expressing α-L-iduronidase and method of treating MPS I by stereotactic injection into the brain of a mammal
US20050143330A1 (en) * 2002-09-30 2005-06-30 Ron Mandel Method for the treatment of Parkinson's Disease
US7605249B2 (en) 2002-11-26 2009-10-20 Medtronic, Inc. Treatment of neurodegenerative disease through intracranial delivery of siRNA
US7618948B2 (en) * 2002-11-26 2009-11-17 Medtronic, Inc. Devices, systems and methods for improving and/or cognitive function through brain delivery of siRNA
US7829694B2 (en) 2002-11-26 2010-11-09 Medtronic, Inc. Treatment of neurodegenerative disease through intracranial delivery of siRNA
US20060018882A1 (en) * 2004-06-21 2006-01-26 Kaemmerer William F Medical devices and methods for delivering compositions to cells
US7732591B2 (en) * 2003-11-25 2010-06-08 Medtronic, Inc. Compositions, devices and methods for treatment of huntington's disease through intracranial delivery of sirna
US7994149B2 (en) 2003-02-03 2011-08-09 Medtronic, Inc. Method for treatment of Huntington's disease through intracranial delivery of sirna
EA200700469A1 (ru) * 2003-02-14 2008-10-30 Байоджен Айдек Ма Инк. Экспрессионная кассета и вектор для временной или постоянной экспрессии экзогенных молекул
US8946151B2 (en) 2003-02-24 2015-02-03 Northern Bristol N.H.S. Trust Frenchay Hospital Method of treating Parkinson's disease in humans by convection-enhanced infusion of glial cell-line derived neurotrophic factor to the putamen
US7963956B2 (en) 2003-04-22 2011-06-21 Antisense Pharma Gmbh Portable equipment for administration of fluids into tissues and tumors by convection enhanced delivery technique
US8927269B2 (en) 2003-05-19 2015-01-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Avian adenoassociated virus and uses thereof
US7186699B2 (en) 2003-06-03 2007-03-06 Cell Genesys, Inc. Method for treating cancer by vector-mediated delivery of one or more anti-angiogenic or pro-apoptotic genes
AU2004245175C1 (en) 2003-06-10 2010-03-18 Biogen Ma Inc. Improved secretion of neublastin
US7261882B2 (en) 2003-06-23 2007-08-28 Reagents Of The University Of Colorado Methods for treating neuropathic pain by administering IL-10 polypeptides
NZ547185A (en) 2003-10-20 2009-03-31 Nsgene As In vivo gene therapy of parkinson's disease
WO2005056807A2 (fr) 2003-12-04 2005-06-23 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, National Institutes Of Health Vecteur viral associe aux adenovirus bovins et leurs utilisations
US20050208090A1 (en) * 2004-03-18 2005-09-22 Medtronic, Inc. Methods and systems for treatment of neurological diseases of the central nervous system
WO2005095450A2 (fr) 2004-03-30 2005-10-13 Nsgene A/S Utilisation therapeutique d'un facteur de croissance, nsg33
WO2006013462A2 (fr) * 2004-07-30 2006-02-09 Nsgene A/S Facteurs de croissance nsg28, nsg30, et nsg32
WO2006029196A1 (fr) * 2004-09-08 2006-03-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, National Institutes Of H Ealth Transcytose de virus adeno-associes
PT1807009E (pt) * 2004-10-05 2015-02-25 Univ California Cânula escalonada
US20060253068A1 (en) * 2005-04-20 2006-11-09 Van Bilsen Paul Use of biocompatible in-situ matrices for delivery of therapeutic cells to the heart
US8283151B2 (en) * 2005-04-29 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Isolation, cloning and characterization of new adeno-associated virus (AAV) serotypes
US7902352B2 (en) * 2005-05-06 2011-03-08 Medtronic, Inc. Isolated nucleic acid duplex for reducing huntington gene expression
US20060257912A1 (en) * 2005-05-06 2006-11-16 Medtronic, Inc. Methods and sequences to suppress primate huntington gene expression
EP2816118B1 (fr) 2005-05-31 2018-10-17 The Regents of the University of Colorado, a body corporate Procédés pour administrer des gènes
US9133517B2 (en) 2005-06-28 2015-09-15 Medtronics, Inc. Methods and sequences to preferentially suppress expression of mutated huntingtin
US20080280843A1 (en) * 2006-05-24 2008-11-13 Van Bilsen Paul Methods and kits for linking polymorphic sequences to expanded repeat mutations
CA2619882C (fr) * 2005-08-23 2015-05-26 The Regents Of The University Of California Canule resistant au reflux et systeme d'administration chronique d'agents therapeutique active par convection
US20070132284A1 (en) * 2005-12-06 2007-06-14 Albert Ekladyous Headliner mounted center high mount stop lamp (chmsl)
ES2352205T3 (es) 2005-12-14 2011-02-16 Licentia Ltd. Usos de una proteína del factor neurotrófico.
AU2007219615B2 (en) 2006-03-03 2013-11-28 Promis Neurosciences Inc. Methods and compositions to treat and detect misfolded-SOD1 mediated diseases
PL2019683T5 (pl) 2006-04-25 2022-12-05 The Regents Of The University Of California Podawanie czynników wzrostu do leczenia zaburzeń OUN
JP2009535360A (ja) * 2006-04-26 2009-10-01 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 高分子量神経治療薬の対流増加送達のための組成物および方法
US9273356B2 (en) 2006-05-24 2016-03-01 Medtronic, Inc. Methods and kits for linking polymorphic sequences to expanded repeat mutations
US7585875B2 (en) * 2006-06-06 2009-09-08 Avigen, Inc. Substituted pyrazolo[1,5-a]pyridine compounds and their methods of use
US20080039415A1 (en) * 2006-08-11 2008-02-14 Gregory Robert Stewart Retrograde transport of sirna and therapeutic uses to treat neurologic disorders
WO2008033285A2 (fr) * 2006-09-15 2008-03-20 The Trustees Of Culumbia University In The City Of New York Délivrance d'arn à double brin dans le système nerveux central
US9375440B2 (en) * 2006-11-03 2016-06-28 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity
US8324367B2 (en) 2006-11-03 2012-12-04 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity
US7819842B2 (en) * 2006-11-21 2010-10-26 Medtronic, Inc. Chronically implantable guide tube for repeated intermittent delivery of materials or fluids to targeted tissue sites
US7988668B2 (en) * 2006-11-21 2011-08-02 Medtronic, Inc. Microsyringe for pre-packaged delivery of pharmaceuticals
US20080171906A1 (en) * 2007-01-16 2008-07-17 Everaerts Frank J L Tissue performance via hydrolysis and cross-linking
EP2131841B1 (fr) 2007-01-30 2012-08-01 Avigen, Inc. Procédés de traitement de la douleur aiguë
EP1970001B1 (fr) * 2007-03-16 2014-07-23 Brainlab AG Cathéter doté d'un capteur de pression
US20090036395A1 (en) * 2007-04-26 2009-02-05 Davidson Beverly L Rna interference suppression of neurodegenerative diseases and methods of use thereof
PL2152346T3 (pl) * 2007-05-17 2016-04-29 Medgenesis Therapeutix Inc Cewnik do konwekcyjnie zwiększonej podaży leku z wyjmowanym usztywniającym elementem
AU2008260103B2 (en) 2007-05-31 2014-04-03 University Of Iowa Research Foundation Reduction of off-target RNA interference toxicity
WO2009018275A1 (fr) * 2007-07-30 2009-02-05 University Of Rochester Adénosine et ses substances mimétiques, modulateurs, inhibiteurs de transport et agonistes de récepteur en tant qu'outil thérapeutique pour remplacer ou améliorer l'efficacité d'une stimulation du cerveau profond
WO2009034127A1 (fr) * 2007-09-12 2009-03-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation du gène cyp46a1 pour le traitement de la maladie d'alzheimer
FI20070808A0 (fi) 2007-10-25 2007-10-25 Mart Saarma GDNF:n silmukointivariantit ja niiden käytöt
FI20080326A0 (fi) 2008-04-30 2008-04-30 Licentia Oy Neurotroofinen tekijä MANF ja sen käytöt
WO2009137630A1 (fr) * 2008-05-07 2009-11-12 Wintherix Llc Procédés pour identifier des composés qui modulent la signalisation wnt dans des cellules cancéreuses
EP2389191A2 (fr) 2009-01-23 2011-11-30 NsGene A/S Expression des neuropeptides dans les cellules des mammiféres
JP2012516357A (ja) 2009-01-29 2012-07-19 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 神経学的障害を治療するための皮質全体に亘る高レベルの治療薬の分布方法
HRP20212024T1 (hr) 2009-05-02 2022-04-01 Genzyme Corporation Genska terapija za neurodegenerativne poremećaje
US8734809B2 (en) 2009-05-28 2014-05-27 University Of Massachusetts AAV's and uses thereof
WO2011025836A1 (fr) * 2009-08-25 2011-03-03 The Regents Of The University Of California Placement optimisé d'une canule pour administration d'agents thérapeutiques au cerveau
EP2498825B1 (fr) 2009-11-09 2017-03-29 Genepod Therapeutics Ab NOUVEAU VECTEUR VIRAL POUR LA SYNTHESE DE DOPA CONTINUÉE ET & xA;SPECIFIQUE AUX NEURONES IN VIVO
EP2558154B1 (fr) 2010-04-16 2020-06-17 ClearPoint Neuro, Inc. Systèmes chirurgicaux pour irm comprenant des canules chirurgicales compatibles avec l'irm pour le transfert d'une substance à et/ou provenant d'un patient
AU2011307488B2 (en) 2010-10-01 2015-08-20 Hoba Therapeutics Aps Use of meteorin for the treatment of allodynia, hyperalgesia, spontaneous pain and phantom pain
WO2012094511A2 (fr) 2011-01-05 2012-07-12 Rhode Island Hospital Compositions et procédés destinés au traitement de lésion ou de maladie orthopédique
WO2012109667A1 (fr) 2011-02-12 2012-08-16 University Of Iowa Research Foundation Composés thérapeutiques
US20120220648A1 (en) * 2011-02-24 2012-08-30 National Taiwan University Hospital Method of Treating Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency Using Adeno-Associated Virus (AAV)-AADC Vector
CN103476399A (zh) * 2011-04-18 2013-12-25 独立行政法人国立精神·神经医疗研究中心 药剂递送粒子及其制造方法
US20130035660A1 (en) 2011-08-01 2013-02-07 Alcyone Lifesciences, Inc. Multidirectional microfluidic drug delivery devices with conformable balloons
CN104105501B (zh) 2011-09-05 2017-10-20 霍巴治疗公司 异常性疼痛、痛觉过敏、自发性疼痛、和幻痛的治疗
CA2869984C (fr) 2012-04-10 2021-08-17 The Regents Of The University Of California Conjugues lipides-peptides bipolymeriques et leurs nanoparticules
EP2884972A4 (fr) * 2012-08-17 2016-05-11 Teva Pharma Formulation parentérale de rasagiline
CA3123066A1 (en) 2012-12-18 2014-06-26 Alcyone Lifesciences, Inc. Systems and methods for reducing or preventing backflow in a delivery system
US9463186B2 (en) 2013-04-15 2016-10-11 Northwestern University Treatment for dopaminergic disorders
BR112015028605A8 (pt) 2013-05-15 2019-12-24 Univ Minnesota uso de uma composição e uso de um imunossupressor e uma composição
WO2014191630A2 (fr) 2013-05-28 2014-12-04 Helsingin Yliopisto Modèle animal non humain codant pour un gène manf non fonctionnel
AU2014281682B2 (en) 2013-06-17 2019-03-21 Alcyone Therapeutics, Inc. Methods and devices for protecting catheter tips and stereotactic fixtures for microcatheters
ES2738298T3 (es) 2013-07-31 2020-01-21 Alcyone Lifesciences Inc Sistemas y métodos de suministro de fármacos, tratamiento y monitoreo
US9891296B2 (en) 2013-09-13 2018-02-13 MRI Interventions, Inc. Intrabody fluid transfer devices, systems and methods
EP3046586B1 (fr) 2013-09-19 2021-07-07 The Regents of The University of California Procédés de traitement au moyen de conjugués bis-polymères peptide-lipide et de leurs nanoparticules
CN106661591B (zh) 2014-05-02 2021-10-29 建新公司 用于视网膜及cns基因疗法的aav载体
RU2711147C2 (ru) 2014-05-20 2020-01-15 Юниверсити Оф Айова Рисерч Фаундейшн Терапевтические соединения для лечения болезни хантингтона
WO2016030748A1 (fr) * 2014-08-25 2016-03-03 Nippon Kayaku Kabushiki Kaisha Délivrance améliorée par convection de micelles chargées en sn-38 contre une tumeur cérébrale
CN107073051B (zh) * 2014-10-21 2021-08-24 马萨诸塞大学 重组aav变体及其用途
SG11201703148TA (en) 2014-11-05 2017-05-30 Voyager Therapeutics Inc Aadc polynucleotides for the treatment of parkinson's disease
WO2016118902A1 (fr) 2015-01-22 2016-07-28 Brown University Contrôle de cellules excitables peu invasif et dépendant de l'activité
US10806396B2 (en) 2015-01-26 2020-10-20 Alcyone Lifesciences, Inc. Drug delivery methods with tracer
LT3256594T (lt) * 2015-02-10 2022-02-10 Genzyme Corporation Patobulintas virusinių dalelių tiekimas į dryžuotąjį kūną ir smegenų žievę
CN106190949B (zh) * 2015-05-08 2020-04-10 上海微创心通医疗科技有限公司 一种干态动物源性胶原纤维组织材料及其制备方法和生物假体
CA2992511A1 (fr) 2015-08-03 2017-02-09 Myodopa Limited Synthese et regulation systemiques de la l-dopa
CA2998500A1 (fr) 2015-09-23 2017-03-30 Sangamo Therapeutics, Inc. Represseurs htt et leurs utilisations
JP2019502473A (ja) 2016-01-04 2019-01-31 アルキオーネ・ライフサイエンシズ・インコーポレイテッドＡｌｃｙｏｎｅ Ｌｉｆｅｓｃｉｅｎｃｅｓ， Ｉｎｃ． 脳卒中を治療するための方法および装置
EP3393571B1 (fr) 2016-02-17 2024-03-06 ClearPoint Neuro, Inc. Ensembles de transfert de fluide chirurgicaux intracorporels avec une longueur de canule à pointe d'aiguille exposée réglable, systèmes associés et procédés
BR112018071156A2 (pt) 2016-04-15 2019-03-12 The Trustees Of The University Of Pennsylvania terapia de gene para tratar mucopolissacaridose tipo ii
EP4273248A3 (fr) 2016-05-20 2024-01-10 Braingene AB Domaines de déstabilisation pour la stabilisation conditionnelle d'une protéine
CA3035522A1 (fr) 2016-08-30 2018-03-08 The Regents Of The University Of California Procedes de ciblage et d'administration biomedicaux, et dispositifs et systemes pour la mise en ƒuvre de ceux-ci
KR20190075964A (ko) 2016-10-13 2019-07-01 유니버시티 오브 매사추세츠 Aav 캡시드 설계
CA3043635A1 (fr) 2016-12-01 2018-06-07 Sangamo Therapeutics, Inc. Modulateurs de tau et procedes et compositions pour leur administration
US10898585B2 (en) 2017-04-14 2021-01-26 Ptc Therapeutics .Inc. Gene therapy for AADC deficiency
JP7246729B2 (ja) 2017-05-18 2023-03-28 国立大学法人京都大学 脊髄小脳変性症３６型の予防又は治療用組成物
JOP20190269A1 (ar) 2017-06-15 2019-11-20 Voyager Therapeutics Inc بولي نوكليوتيدات aadc لعلاج مرض باركنسون
JP7229989B2 (ja) 2017-07-17 2023-02-28 ボイジャー セラピューティクス インコーポレイテッド 軌道アレイガイドシステム
US12098385B2 (en) 2017-09-20 2024-09-24 The Regents Of The University Of California Gene therapy strategy to restore cardiac electrical and structural function in arrhythmogenic right ventricular cardiomyopathy
EP3684422B1 (fr) 2017-09-20 2023-12-20 The Regents of the University of California Connexine-43 pour la thérapie génique destinée à restaurer une fonction électrique et cardiaque, et la structure cardiaque, dans une cardiomyopathie ventriculaire droite arythmogène
KR20200104852A (ko) 2017-09-22 2020-09-04 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 Ii형 점액다당류증의 치료를 위한 유전자 요법
PE20201501A1 (es) 2018-01-12 2020-12-29 Bristol Myers Squibb Co Oligonucleotidos antisentido que actuan sobre alfa-sinucleina, y usos de estos
US11253237B2 (en) 2018-05-09 2022-02-22 Clearpoint Neuro, Inc. MRI compatible intrabody fluid transfer systems and related devices and methods
EP3781074A1 (fr) 2018-05-09 2021-02-24 ClearPoint Neuro, Inc. Systèmes de transfert de liquide intracorporel compatible mri et dispositifs et procédés associés
SG11202011296VA (en) * 2018-05-15 2020-12-30 Voyager Therapeutics Inc Compositions and methods for the treatment of parkinson's disease
US11684750B2 (en) 2019-10-08 2023-06-27 Clearpoint Neuro, Inc. Extension tube assembly and related medical fluid transfer systems and methods
KR20240006581A (ko) 2021-05-06 2024-01-15 호바 세라퓨틱스 에이피에스 화학요법-유도된 신경병성 통증의 예방 및 치료
AU2022405685A1 (en) 2021-12-10 2024-07-11 Hoba Therapeutics Aps Treatment of nociceptive pain

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US4916151A (en) * 1985-12-05 1990-04-10 Merrell Dow Pharmaceuticals Inc. Method of treating parkinson's syndrome
JP2514913B2 (ja) * 1987-10-19 1996-07-10 忠三 岸本 ヒトｂｃｄｆを含有する神経系障害治療剤
DE3841955A1 (de) * 1987-12-31 1989-07-13 Asta Pharma Ag Synergistische kombination von decarboxylasehemmern und l-dopa-pellets
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5766948A (en) * 1993-01-06 1998-06-16 The Regents Of The University Of California Method for production of neuroblasts
WO1995005864A1 (fr) * 1993-08-27 1995-03-02 Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Systeme d'administration de medicament amelioree par convexion
EP0755454B1 (fr) 1994-04-13 2008-02-13 The Rockefeller University Transmission par virus adenoassocie d'adn a des cellules du systeme nerveux
US5658785A (en) 1994-06-06 1997-08-19 Children's Hospital, Inc. Adeno-associated virus materials and methods
US6103226A (en) 1994-08-12 2000-08-15 Arch Development Corporation Genetically engineered cells that produce produce L. Dopa
US5599706A (en) * 1994-09-23 1997-02-04 Stinchcomb; Dan T. Ribozymes targeted to apo(a) mRNA
US5667158A (en) 1995-05-23 1997-09-16 Glaxo Wellcome Inc. Automated blend reclaim system for pharmaceutical tablet compression machine
US5677158A (en) 1995-06-07 1997-10-14 Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US6004797A (en) 1995-11-09 1999-12-21 Avigen, Inc. Adenovirus helper-free recombinant AAV Virion production
AU3591597A (en) * 1996-06-28 1998-01-21 Regents Of The University Of California, The Transformation and genetherapy of cells of the inner ear
JPH1113A (ja) 1997-06-12 1999-01-06 Mitsubishi Agricult Mach Co Ltd 乗用農機
ES2324540T3 (es) * 1998-05-27 2009-08-10 Genzyme Corporation Vectores aav para la fabricacion de medicamentos para la administracion potenciada por conveccion.

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1997 DEJESUS ONOFRE T ET AL: "Evaluation of fluorinated m-tyrosine analogs as PET imaging agents of dopamine nerve terminals: Comparison with 6-fluorodopa." Database accession no. PREV199799523020 XP002221587 & JOURNAL OF NUCLEAR MEDICINE, vol. 38, no. 4, 1997, pages 630-636, ISSN: 0161-5505 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1997 JORDAN SHAUN ET AL: "6-(18F)fluoro-L-m-tyrosine: Metabolism, positron emission tomography kinetics, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridi ne lesions in primates." Database accession no. PREV199799506769 XP002221586 & BRAIN RESEARCH, vol. 750, no. 1-2, 1997, pages 264-276, ISSN: 0006-8993 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1997 WAHL L M ET AL: "The use of (18F)6-fluoro-L-m-tyrosine in clinical studies of movement disorders." Database accession no. PREV199799626183 XP002221589 & JOURNAL OF NUCLEAR MEDICINE, vol. 38, no. 5 SUPPL., 1997, page 274P 44th Annual Meeting of the Society of Nuclear Medicine;San Antonio, Texas, USA; June 1-5, 1997 ISSN: 0161-5505 *
DATABASE MEDLINE [Online] NAHMIAS ET AL: "A probe for intracerebral aromatic amino-acid decarboxylase activity: distribution and kinetics of [18FÜ6-fluoro-L-m-tyrosine in the human brain" Database accession no. NLM7651447 XP002221588 & MOVEMENT DISORDERS: OFFICIAL JOURNAL OF THE MOVEMENT DISORDER SOCIETY, vol. 10, 1995, pages 298-304, *
See also references of WO9961066A2 *

Also Published As

Publication number Publication date
US20180243249A1 (en) 2018-08-30
WO1999061066A3 (fr) 2000-05-04
ES2324540T3 (es) 2009-08-10
ATE316576T1 (de) 2006-02-15
WO1999061066A2 (fr) 1999-12-02
US6309634B1 (en) 2001-10-30
US20160256534A1 (en) 2016-09-08
ES2326893T3 (es) 2009-10-21
US20130101510A1 (en) 2013-04-25
US20020141980A1 (en) 2002-10-03
ATE431418T1 (de) 2009-05-15
US20100104537A1 (en) 2010-04-29
US20050180955A1 (en) 2005-08-18
DE69929600D1 (de) 2006-04-13
US6953575B2 (en) 2005-10-11
DE69929600T2 (de) 2006-09-07
ES2257051T3 (es) 2006-07-16
EP1080202B1 (fr) 2006-01-25
DE69940899D1 (de) 2009-06-25
US7534613B2 (en) 2009-05-19
ATE435917T1 (de) 2009-07-15
JP2002516295A (ja) 2002-06-04
CA2329259C (fr) 2003-08-05
PT1080202E (pt) 2006-05-31
CA2329259A1 (fr) 1999-12-02
JP2006298926A (ja) 2006-11-02
US9492415B2 (en) 2016-11-15
DE69941100D1 (de) 2009-08-20
WO1999061066A9 (fr) 2000-03-30
US8309355B2 (en) 2012-11-13

Similar Documents

Publication Publication Date Title
US20180243249A1 (en) Methods of treating parkinson's disease using viral vectors
Bankiewicz et al. Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach
CA2457057C (fr) Compositions et procedes destines au traitement des maladies neurodegeneratives
US20220347319A1 (en) Methods for distributing high levels of therapeutic agent throughout the cortex to treat neurological disorders
AU2002326163A1 (en) Compositions and methods for treating neurodegenerative diseases
US20060073119A1 (en) Methods for treating neurodegenerative disorders
WO2005037211A2 (fr) Methodes et compositions pour le traitement de maladies neurologiques
EP1621625B1 (fr) Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
CA2425820C (fr) Apport de vecteurs viraux adeno-associes (aav) ameliore par convection
EP2558113A2 (fr) Compositions thérapeutiques de serca2 et méthodes d'utilisation

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20001218

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIC1 Information provided on ipc code assigned before grant

Free format text: 7C 12N 15/54 A, 7C 12N 15/60 B, 7A 61K 48/00 B, 7A 61K 51/00 B, 7A 61K 51/04 B

17Q First examination report despatched

Effective date: 20021212

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 61P 25/16 B

Ipc: 7A 61K 31/195 B

Ipc: 7A 61K 38/51 B

Ipc: 7A 61K 51/04 B

Ipc: 7A 61K 51/00 B

Ipc: 7A 61K 48/00 B

Ipc: 7C 12N 15/60 B

Ipc: 7C 12N 15/54 A

RTI1 Title (correction)

Free format text: CONVECTION-ENHANCED DELIVERY OF AAV VECTORS ENCODING AADC

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: THE REGENTS OF THE UNIVERSITYOF CALIFORNIA

Owner name: AVIGEN, INC.

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060125

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060125

Ref country code: CH

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060125

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060125

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 69929600

Country of ref document: DE

Date of ref document: 20060413

Kind code of ref document: P

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060425

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060425

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060526

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060531

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Effective date: 20060327

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20060401435

Country of ref document: GR

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2257051

Country of ref document: ES

Kind code of ref document: T3

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20061026

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060526

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060125

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 17

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 18

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 19

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20180329

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20180314

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20180601

Year of fee payment: 20

Ref country code: PT

Payment date: 20180516

Year of fee payment: 20

Ref country code: DE

Payment date: 20180515

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20180514

Year of fee payment: 20

Ref country code: IT

Payment date: 20180522

Year of fee payment: 20

Ref country code: GR

Payment date: 20180411

Year of fee payment: 20

Ref country code: FR

Payment date: 20180411

Year of fee payment: 20

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 69929600

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: MK

Effective date: 20190525

REG Reference to a national code

Ref country code: GB

Ref legal event code: PE20

Expiry date: 20190525

Ref country code: BE

Ref legal event code: MK

Effective date: 20190526

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20190531

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20190525

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20220126

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20190527